Biomarkers For Clinical Diagnosis Research Articles

Electrochemical Impedimetric Platform Based on Con A@MIL-101 for Glycoprotein Detection.

An electrochemical impedimetric biosensing platform with lectin as a molecular recognition element has been established for the sensitive detection of glycoproteins, a class of important biomarkers in clinical diagnosis. One of the representative metal-organic framework materials, MIL-101(Cr)-NH2, was utilized as the supporting matrix, and its amino groups served as the anchors to immobilize the lectins of concanavalin A (Con A), constituting Con A@MIL-101(Cr)-NH2 for the determination of invertase (INV) as a model glycoprotein. The Con A concentration, immobilization time, and incubation time with INV were optimized. Under the optimal conditions, the degree of impedance increase was linearly proportional to the logarithm of INV concentration between 1.0 × 10-16 and 1.0 × 10-11 M, affording a limit of detection as low as 3.98 × 10-18 M. Good specificity, stability, reproducibility, and repeatability were demonstrated for the fabricated biosensing platform. Moreover, real mouse serum samples were spiked with different concentrations of INV. Excellent recoveries were obtained, which demonstrated the biosensing platform's capability of analyzing glycoproteins within a complex matrix.

Abstract 2410: Comparative genomic profiling of unresectable NSCLC patients in the U.S. and China using a globally harmonized liquid biopsy assay platform

Abstract Background: Non-Small Cell Lung Cancer (NSCLC), accounting for over 80% of all lung cancers, benefits from targeted therapies based on genetic tests like EGFR, KRAS, ALK, and ERBB2. While prior studies focused on molecular characteristics through tissue biopsies, limited research has explored NSCLC molecular profiles via liquid biopsy, especially across different human races. This study presents a comprehensive genomic profiling analysis of unresectable NSCLC patients in the U.S. and China, using a globally harmonized liquid biopsy assay. Methods: As part of Predicine's Phoenix Program, a global molecular biomarkers screening initiative, 61 patients in the U.S. and 352 patients in China with unresectable advanced NSCLC were enrolled. All were treatment-naïve or recurred after 1st-line targeted therapies. Blood samples (10ml) were tested using PredicineCARE, an NGS-based liquid biopsy assay, to profile somatic mutations, copy number variations, and gene fusions. Results: The assay, validated in both regions using the same reference materials, achieved a Limit of Detection (LOD) of 0.25% mutation allele frequency, with a positive predictive value exceeding 99%. Profiling NSCLC patients in the U.S. and China revealed common genes like TP53, CDKN2A, EGFR, KRAS, RB1, and PIK3CA. TP53 and PIK3CA variations showed equivalent prevalence. However, EGFR variations were significantly higher in China (p<0.05), while CDKN2A (p<0.001), KRAS (p<0.01), and RB1 (p<0.01) variations were notably higher in the U.S. Conclusions: This study unveils the mutational landscape of advanced NSCLC through liquid biopsy. Unique prevalence patterns between U.S. and China cohorts suggest novel biomarkers for clinical diagnosis and provide insights for targeted therapy mechanism studies. Citation Format: Haoran Tang, Cancan Jia, Feng Xie, Yue Zhang, Xiaoxi Dong, Yong Huang, Shading Jia. Comparative genomic profiling of unresectable NSCLC patients in the U.S. and China using a globally harmonized liquid biopsy assay platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2410.

Abstract 5015: Genomic profiling of colorectal cancer - insights from liquid biopsy comparisons between U.S. and China cohorts

Abstract Background: Molecular characteristics play a pivotal role in cancer diagnosis, treatment selection, and disease monitoring across various tumor types. While previous research has elucidated the molecular classification of colorectal cancer using tissue NGS, limited investigation has explored the molecular profile of colorectal cancer through liquid biopsy, especially across diverse human races. This study presents a comprehensive genomic profiling analysis of colorectal cancer patients, using a globally harmonized liquid biopsy assay to compare patient cohorts from both the U.S. and China. Methods: The prospective study is part of Predicine’s Phoenix Program, a global molecular biomarker screening initiative across multiple solid tumors. At present, the study has enrolled 57 patients in the U.S. and 62 patients in China, all presenting with advanced colorectal cancer. A 10ml blood sample was collected from each patient and tested using PredicineCARE, an NGS-based liquid biopsy assay, to profile somatic mutations, copy number variations, and gene fusions among these patients. Results: The assay, validated in both the U.S. and China using the same reference materials, achieved a LOD of 0.25% mutation allele frequency, with a positive predictive value exceeding 99%. Subsequently, the assay was applied to profile molecular aberrations in colorectal patients in both the U.S. and China. Among U.S. patients, the most commonly altered genes were APC (60%), KRAS (56%), TP53 (54%), MYC (25%), PIK3CA (16%), and EGFR (14%). In Chinese patients, the predominant altered genes were TP53 (48%), APC (42%), KRAS (32%), GNAS (21%), EGFR (16%), and PIK3CA (11%). The prevalence of GNAS variations was significantly higher in China than in the U.S. (p < 0.05), while the prevalence of APC (p < 0.05), KRAS (p < 0.01), and MYC (p < 0.001) variations was significantly higher in the U.S. than in China. Conclusions: This study reveals the extensive mutational landscape in advanced colorectal cancer patients through liquid biopsy. Distinct prevalence patterns in certain genes between the U.S. and China cohorts offer novel biomarkers for clinical diagnosis and provide insights for targeted therapy mechanism studies. Citation Format: Haoran Tang, Cancan Jia, Feng Xie, Yue Zhang, Xiaoxi Dong, Yong Huang, Shidong Jia. Genomic profiling of colorectal cancer - insights from liquid biopsy comparisons between U.S. and China cohorts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5015.

Identification of potential serum biomarkers associated with HbA1c levels in Indian type 2 diabetic subjects using NMR-based metabolomics

BackgroundThe prevalence of type 2 diabetes mellitus (T2DM), a progressive metabolic disorder characterized by chronic hyperglycemia and the development of insulin resistance, has increased globally, with worrying statistics coming from children, adolescents, and young adults from developing countries like India. Here, we investigated unique circulating metabolic signatures associated with prediabetes and T2DM in an Indian cohort using NMR-based metabolomics. Materials and methodsThe study subjects included healthy volunteers (N = 101), prediabetic subjects (N = 75), and T2DM patients (N = 108). Serum metabolic profiling was performed using 1H NMR spectroscopy and major perturbed metabolites were identified by multivariate analysis and receiver operating characteristic (ROC) modules. ResultsOf the 36 aqueous abundant metabolites, 24 showed a statistically significant difference between healthy volunteers, prediabetics, and established T2DM subjects. On performing multivariate ROC curve analysis with 5 commonly dysregulated metabolites (namely, glucose, pyroglutamate, o-phosphocholine, serine, and methionine) in prediabetes and T2DM, AUC values obtained were 0.96 (95 % confidence interval (CI) = 0.93, 0.98) for T2DM; and 0.88 (95 % CI = 0.81, 0.93) for prediabetic subjects, respectively. ConclusionWe propose that the identified metabolite panel can be used in the future as a biomarker for clinical diagnosis, patient surveillance, and for predicting individuals at risk for developing diabetes.

Magnetic graphene oxide and vertically-ordered mesoporous silica film for universal and sensitive homogeneous electrochemiluminescence aptasensor platform

Herein, we demonstrated a convenient and highly sensitive homogeneous electrochemiluminescence (ECL) aptasensor platform by combination of magnetic composite probe and vertically-ordered mesoporous silica films (VMSF). Magnetic composite probe composed of biorecognition element (target-specific aptamer), ECL signal reporter (Ru(bpy)32+) and large-scale magnetic graphene oxide (M−GO), can be prepared by simple incubation, magnetic separation and redispersion prior to use. High-affinity and specific binding between target and aptamer can detach aptamer from M−GO and simultaneously release the slight amount of Ru(bpy)32+ into a solution, which can be sensitively detected by VMSF modified indium tin oxide (VMSF/ITO) due to the excellent electrostatic preconcentration capacity of VMSF. In this sensing system, detectable signal of Ru(bpy)32+ can be remarkably amplified by VMSF/ITO and meanwhile background signal can be reduced by the adsorption capacity of M−GO and size exclusion property of VMSF/ITO, effectively promoting the analytical performance of the present homogeneous ECL aptasensor. The applicability and universality of this sensing platform was confirmed by employing alpha fetoprotein (AFP) and prostate specific antigen (PSA) as the target models and their detection limits were 0.08 pg/mL and 9.6 pg/mL, respectively. Furthermore, the designed homogeneous ECL aptasensor platform is label-free, immobilization-free, low-cost, and free of additional electrode modification and sample pretreatment procedures, rendering a simple sensing strategy for quantitative determination of various biomarkers in clinical diagnosis by adapting the specific aptamer.

Endogenously Gated DNA Walking Machine for Prescreened MicroRNA Detection in Extracellular Vesicles.

Tumor-derived extracellular vesicle (T-EV) microRNAs have been investigated as promising biomarkers in clinical diagnosis as well as disease progression monitoring. However, the expression profiles of microRNA in different tissues vary widely, the precise monitoring of microRNA levels in EVs originating from diseased tissues is susceptible to background interference, thus remains a challenge. Conventional assays require extensive processing, such as EV isolation and even sample lysis, which is both slow and laborious, and the cumbersome pretreatment could spoil the downstream analysis. To address this issue, we developed a generalizable strategy for T-EVs-selective activation and therefore specific amplified microRNA imaging. The conditional signal amplification is established by integrating a traditional DNA walker system with endogenously activated motif to achieve sensitized microRNA imaging in T-EVs. The preorganized endogenous activation with additional sensing criteria narrowed the scope against the complex specimens, and the amplified sensing with reduced off-target signals was supposed to be sensitive to monitor the tiny changes of microRNA expression during the disease course, which holds great potential for accurate diagnosis and prognosis.

Liquid biopsy-based comprehensive genomic profiling to reveal mutational landscape in real-world patients with gastrointestinal cancer.

735 Background: Molecular characteristics hold substantial clinical significance in cancer. Previous research has elucidated the molecular classification of gastrointestinal (GI) cancers, leveraging biomarkers like gene alterations, MSI, EBV status, and chromosomal instability. However, most of these studies relied on tissue biopsies. Limited investigations have been documented regarding the characterization of molecular profiles through liquid biopsy. Here we present a comprehensive genomic profiling study conducted on advanced GI cancer patients using blood samples. Methods: This prospective study is a part of a global initiative focused on molecular biomarker screening across various solid tumors. Here we report the early result of 106 unresectable GI cancer patients, encompassing those with advanced disease or experiencing relapse after prior treatments. The cohort comprises 66 colorectal cancer (CRC) patients, 34 gastric cancer (GC) patients, and 6 esophageal cancer (EC) patients. Each patient contributed a 10-mL blood sample for comprehensive circulating tumor DNA (ctDNA) analysis. The study employed PredicineCARE, a 152-gene, NGS-based liquid biopsy assay, to profile somatic gene variations within this patient population. Results: The study revealed a total of 1,597 somatic mutations, with 1,077 occurring in CRC, 448 in GC, and 72 in EC. Additionally, 722 gene copy number variants were identified, with 288 in CRC, 351 in GC, and 83 in EC patients. In CRC, the most frequently altered genes (prevalence > 20%) included TP53 (58%), APC (45%), KRAS (33%), and BRCA2 (22%). Conversely, in GC, the most altered genes were TP53 (59%), TERT (35%), FAT1 (29%), ARID1A (26%), and FGFR4 (24%). Among the 6 EC patients, 3 carried TP53 variations, with variations in APC (N=2), ERCC5 (N=2), FAT1 (N=2), and EGFR (N=1) also observed. The mutation burden in CRC (median 3.73 mut/Mb) and GC (median 3.57 mut/Mb) displayed no significant differences. However, the mutation burden in CRC was significantly higher than that in EC (2.24 mut/Mb) (p=0.04). A similar pattern was observed between GC and EC but did not reach statistical significance (p=0.10) due to limitations in cohort size. Furthermore, the assay identified patients carrying both germline and somatic mutations related to homologous recombination deficiency (HRD). In CRC patients, 5 had MLH1 germline mutations, 6 had MSH2 mutations (5 were germline), 4 had MSH6 germline mutations, 2 had PMS2 somatic mutations, and 1 had an EPCAM germline mutation. While in GC patients, 2 had MLH1 germline mutations, 1 had an MSH6 mutation, and 1 had a PMS2 somatic mutation. Conclusions: This study elucidated the comprehensive mutational landscape of advanced gastrointestinal cancer through liquid biopsy, thereby contributing novel biomarkers for clinical diagnosis and facilitating targeted therapy mechanism investigations.

Development and validation of a novel lysosome-related LncRNA signature for predicting prognosis and the immune landscape features in colon cancer

Lysosomes are essential components for managing tumor microenvironment and regulating tumor growth. Moreover, recent studies have also demonstrated that long non-coding RNAs could be used as a clinical biomarker for diagnosis and treatment of colorectal cancer. However, the influence of lysosome-related lncRNA (LRLs) on the progression of colon cancer is still unclear. This study aimed to identify a prognostic LRL signature in colon cancer and elucidated potential biological function. Herein, 10 differential expressed lysosome-related genes were obtained by the TCGA database and ultimately 4 prognostic LRLs for conducting a risk model were identified by the co-expression, univariate cox, least absolute shrinkage and selection operator analyses. Kaplan–Meier analysis, principal-component analysis, functional enrichment annotation, and nomogram were used to verify the risk model. Besides, the association between the prognostic model and immune infiltration, chemotherapeutic drugs sensitivity were also discussed in this study. This risk model based on the LRLs may be promising for potential clinical prognosis and immunotherapeutic responses related indicator in colon cancer patients.

Ratiometric antifouling electrochemical biosensors based on designed Y-shaped peptide and MXene loaded with Au@ZIF-67 and methylene blue.

Based on the designed inverted Y-shaped peptide and MXene nanocomposite (MXene-Au@ZIF-67), a ratiometric anti-pollution electrochemical biosensor was designed and applied to the detection of biomarkers in serum. Au@ZIF-67 inserted into the interior of MXene can not only prevent the accumulation of MXene but also provide a large amounts of binding sites for capturing biomolecules. A designed multifunctional Y-shaped peptide containing anchoring, antifouling, and recognition sequences was anchored onto MXene-Au@ZIF-67 through Au-S bonds. Electrochemical signal molecules, ferrocenecarboxylic acid (Fc) and methylene blue (MB), were modified to another end of multifunctional peptide and interior of MXene-Au@ZIF-67, respectively, to produce aratiometric electrochemical signal. We selected prostate specific antigen (PSA) as the modelcompound. PSA specifically recognizes and cleaves the recognition segment in the Y-shaped peptide, and the signal of Fc is reduced, while the signal of MB remains unchanged. The ratiometric strategy endows the present biosensorhigh accuracy and sensitivity with adetection limit of 0.85pg/mL. In addition, the sensing surface has good antifouling ability due to the antifouling sequence of the two branching parts of the Y-shaped peptide. More importantly, by replacing the recognition segment of peptides alsoother targetsare accessible, indicating the potential application of the universal detection strategy tothe detection of various biomarkers in clinical diagnosis.

Dual-signal amplification sensing platform based on competitive host-guest cycling recognition process for ultrasensitive detection of thyroglobulin

Dual-signal amplification sensing strategy has been constructed by the competitive host-guest cycling recognition process. Owing to superior photoelectric and photothermal performance of In2Se3 nanoparticles (NPs), the multifunctional probe modified with In2Se3 NPs can generate significantly increased photocurrent and temperature dual signals output during the one-time sensing process. To further achieve signal amplification, with the assistance of supramolecular host-guest recognition between β-cyclodextrin (β-CD) and adamantine (ADA), the energy band-matching TiO2 MOF was introduced to construct In2Se3/TiO2 heterojunction that enhance the separation efficiency of photogenerated electron-hole pairs. Interestingly, we found that quantum well-like interface structure (TiO2/In2Se3/TiO2) can be constructed through competitive host-guest cycling recognition process to continuously amplify the signals. The multiple heterostructures can significantly improve light absorption efficiency, not only effectively enhancing the anode photocurrent signal but also generating a robust photothermal response upon the excitation of near-infrared light irradiation. Based on this, the developed dual-signal amplification sensing platform realized ultrasensitive detection of target thyroglobulin (Tg) with linear range from 1.0 × 10−5 ng/mL to 1 ng/mL (R2 =0.98) and low detection limit 3.3 × 10−6 ng/mL. The strategy unprecedentedly couples In2Se3 NPs and TiO2 MOF to construct dual-signal amplification interfaces through competitive host-guest cycling recognition process, showing great promise for detection of the target biomarkers in clinical diagnosis.

IMMT promotes hepatocellular carcinoma formation via PI3K/AKT/mTOR pathway

Abstract Objectives Hepatocellular carcinoma (HCC) remains a global challenge. Finding new specific targets has significant clinical value for the treatment of hepatocellular carcinoma. In this study, we discovered a new biomarker targeting HCC. At present, the role of Inner Membrane Mitochondrial Protein (IMMT) in various malignant tumors is receiving increasing attention, but its molecular mechanism of action in the malignant process of HCC has not been fully elucidated. This study aims to investigate the key regulatory mechanisms of IMMT in the formation of HCC. Methods Using TCGA data and clinical HCC samples, we first studied the expression of IMMT in HCC tissues and its correlation with malignant prognosis of patients. The regulatory effect of IMMT on HCC was studied by lentivirus infection. In vitro, the effects of IMMT on the proliferation, migration and apoptosis of cells were investigated by CCK8, colony formation assay, transwell and flow cytometry. Consistently, in vivo experiments, the regulation of tumor growth by IMMT was studied by constructing subcutaneous transplanted tumor and liver carcinoma in situ. In terms of mechanism, we predicted and verified the downstream genes of IMMT with the help of string database. Results We found that IMMT was significantly up-regulated in HCC tissues and was significantly positively correlated with poor prognosis of patients. Functionally, we demonstrated that IMMT knockdown significantly inhibited HCC cell proliferation, migration and promoted cell apoptosis in vitro. Similarly, the knockdown of IMMT also significantly weakened the progression of tumors in vivo. In terms of mechanism, we demonstrate for the first time that IMMT can regulate the progression of HCC by influencing the activation of PI3K-AKT-mTOR pathway. Conclusions Collectively, our research findings elucidate the hitherto unexplored important role of the IMMT/PI3K/AKT/mTOR axis in the formation of HCC, and provide a new biomarker for clinical diagnosis and treatment of HCC.

Persistent endothelial dysfunction in post-COVID-19 syndrome and its associations with symptom severity and chronic inflammation

BackgroundPost-COVID-19 syndrome (PCS) is a lingering disease with ongoing symptoms such as fatigue and cognitive impairment resulting in a high impact on the daily life of patients. Understanding the pathophysiology of PCS is a public health priority, as it still poses a diagnostic and treatment challenge for physicians.MethodsIn this prospective observational cohort study, we analyzed the retinal microcirculation using Retinal Vessel Analysis (RVA) in a cohort of patients with PCS and compared it to an age- and gender-matched healthy cohort (n = 41, matched out of n = 204).Measurements and main resultsPCS patients exhibit persistent endothelial dysfunction (ED), as indicated by significantly lower venular flicker-induced dilation (vFID; 3.42% ± 1.77% vs. 4.64% ± 2.59%; p = 0.02), narrower central retinal artery equivalent (CRAE; 178.1 [167.5–190.2] vs. 189.1 [179.4–197.2], p = 0.01) and lower arteriolar-venular ratio (AVR; (0.84 [0.8–0.9] vs. 0.88 [0.8–0.9], p = 0.007). When combining AVR and vFID, predicted scores reached good ability to discriminate groups (area under the curve: 0.75). Higher PCS severity scores correlated with lower AVR (R = − 0.37 p = 0.017). The association of microvascular changes with PCS severity were amplified in PCS patients exhibiting higher levels of inflammatory parameters.ConclusionOur results demonstrate that prolonged endothelial dysfunction is a hallmark of PCS, and impairments of the microcirculation seem to explain ongoing symptoms in patients. As potential therapies for PCS emerge, RVA parameters may become relevant as clinical biomarkers for diagnosis and therapy management.Trial registrationThis study was previously registered at ClinicalTrials (“All Eyes on PCS—Analysis of the Retinal Microvasculature in Patients with Post-COVID-19 Syndrome”. NCT05635552. https://clinicaltrials.gov/ct2/show/NCT05635552).Graphical abstractPersistent endothelial dysfunction in post-COVID-19 syndrome. Acute SARS-CoV-2 infection indirectly or directly causes endotheliitis in patients. N = 41 PCS patients were recruited and retinal vessel analysis was performed to assess microvascular endothelial function. Images of SVA and DVA are illustrative for RVA data analysis. For each PCS patient and healthy cohort, venular vessel diameter of the three measurement cycles was calculated and plotted on a diameter-time curve. Patients exhibited reduced flicker-induced dilation in veins (vFID) measured by dynamic vessel analysis (DVA) and lower central retinal arteriolar equivalent (CRAE) and arteriolar-venular ratio (AVR) and a tendency towards higher central retinal venular equivalent (CRVE) when compared to SARS-CoV-2 infection naïve participants. Created with BioRender.com

P-637 pseudo-targeted metabolomics of follicular fluid reveals ovarian GPD1L-mediated glycerophospholipid metabolism dysfunction in patients with biochemical premature ovarian insufficiency

Abstract Study question Does the metabolic profile of follicular fluid from patients with biochemical premature ovarian insufficiency (bPOI) differ from that of healthy women? Summary answer The metabolic signature of follicular fluid reveals a significant decrease of phosphatidic acid (PA), phosphatidylcholine (PC), lysophosphatidylcholine (lysoPC) and lysophosphatidylethanolamine (lysoPE) in bPOI patients. What is known already Metabolites in follicular fluid have attracted extensive attention as they are important components of the microenvironment during follicular development and oocyte maturation. Studies on follicular fluid metabolomics may enhance the understanding of the pathogenesis of ovarian aging, particularly premature ovarian insufficiency. Although a variety of metabolites have been suggested to be related to cell senescence, their specific roles in regulating follicular atresia remain unclear. Study design, size, duration An observational study of follicular fluid samples collected from 120 women undergoing in vitro fertilization and embryo transfer was conducted. All enrolled women were classified into two groups: one with 60 bPOI patients and the other with 60 healthy controls. Each group was further subdivided into two subgroups (young and aged group, 30 women for each section). Participants/materials, setting, methods The follicular fluids of mature follicles (diameter over 17 millimeters) were isolated and collected for pseudo-targeted metabolic analysis. The intensities of captured metabolites were utilized for further bioinformatic analysis. The correlation between the significant differential metabolites screened and their clinical outcomes were analyzed. In vitro experiments were used to demonstrate the cellular function of mainly regulated signaling pathway. Main results and the role of chance A total of 608 metabolites were identified in 120 follicular fluid samples. There were significant differences in 143 metabolites between the bPOI and healthy controls. As revealed by metabolic pathway analysis, glycerophospholipid metabolism was the most enriched pathway in bPOI follicular fluids. The intensities of several glycerophospholipid-associated metabolites PA, PC, lysoPC and lysoPE were positively correlated with the ovarian reserve indicator anti-Müllerian hormone (AMH), female age, as well as the expression level of glycerol-3-phosphatedehydrogenase 1-like (GPD1L), a key enzyme of this pathway, in granulosa cells. Furthermore, knockdown of GPD1L induced mitochondria dysfunction and granulosa cell apoptosis via GPD1L/PKCε/ATF2/NEAT1 pathway, as well as led to follicular atresia and impaired the quality of oocytes. Limitations, reasons for caution The validity of differential intensified metabolites we detected as biomarkers for clinical diagnosis needs to be confirmed in a large-scale study. And the multiple effects of glycerophospholipid metabolic pathway posed on follicular development need to be study in-depth. Wider implications of the findings Our results suggested that a highly coordinated follicular metabolism exhibits a strong relationship with the quality of follicles. An understanding of the mechanisms underlying ovarian aging will provide a theoretical basis, as well as new ideas for POI diagnosis, treatment and intervention. Trial registration number Not applicable.

High-efficient Pt@COF nanospheres-based electrochemical-chemical-chemical redox cycling for ultrasensitive microRNAs biosensing

MicroRNAs (miRNAs) have been perceived as important regulators in multifarious biological processes as well as potential biomarkers in clinical diagnosis. Exploring high-efficient sensing platforms for tracing miRNAs in complex biological samples is of great vital. Herein, an ultrasensitive and enzyme-free electrochemical biosensor was constructed based on integration of efficiently catalytic electrochemical−chemical−chemical (ECC) redox cycling with target-induced magnetic DNAzyme walker. Employing porous and easily functionalized covalent organic framework (COF) as carriers, Pt@COF nanospheres (Pt@COF NSs) were facilely prepared by in-situ reduction of platinum precursors in the nanopores-structure of COF. This not only addressed the inevitable migration of pure Pt nanoparticles, but endowed Pt@COF NSs with desirable stability and excellent catalytic activity to boost ECC redox cycling for effective signal enhancement. On this basis, a target-induced magnetic DNAzyme walker was obediently induced to recognize, separate and convert low-abundant miRNA-21 targets into plentiful output DNA chains for accumulated signal amplification. Finally, the as-prepared robust biosensor manifested highly sensitive and selective determination of miRNA-21 with a wide detection range from 100 aM to 10 pM and a low detection limit of 47.5 aM. Meanwhile, the credible detectability and anti-interference were also demonstrated in serum and cell samples, indicating its promising application toward diseases warning.

Whole transcriptome sequencing analysis of blood plasma-derived exosomes from immune-related hearing loss

BackgroundEarly diagnosis of immune-related hearing loss and timely treatment can prevent structural damage to the inner ear and contribute to hearing retention. Exosomal miRNAs, lncRNAs and proteins have great prospects as novel biomarkers for clinical diagnosis. Our study aimed to investigate the molecular mechanisms of exosomes or exosomal ceRNA regulatory networks in immune-related hearing loss. MethodsAn immune-related hearing loss mice model was constructed by injection with inner ear antigen, and then the blood plasma samples of the mice were collected for exosomes isolation by ultra-centrifugation. Subsequently, the different exosomes were sent for whole transcriptome sequencing using Illumina platform. Finally, a ceRNA pair was chosen for validation by RT-qPCR and dual luciferase reporter gene assay. ResultsThe exosomes were successfully extracted from the blood samples of the control and the immune-related hearing loss mice. After sequencing, 94 differentially expressed (DE) lncRNAs, 612 DEmRNAs, and 100 DEmiRNAs were found in the immune-related hearing loss-associated exosomes. Afterwards, ceRNA regulatory networks consisting of 74 lncRNAs, 28 miRNAs and 256 mRNAs were proposed, and the genes in the ceRNA regulatory networks were significantly enriched in 34 GO terms of biological processes and 9 KEGG pathways. Finally, Gm9866 and Dusp7 were significantly up-regulated, while miR-185-5p level was declined in the exosomes from immune-related hearing loss, and Gm9866, miR-185-5p and Dusp7 interacted with each other. ConclusionsGm9866-miR-185-5p-Dusp7 was confirmed to be closely correlated with the occurrence and progression of immune-related hearing loss.

A novel photoelectrochemical strategy for sequence-spot bispecific analysis of N6-methyladenosine modification based on proximity ligation-triggered cascade amplification

N6-methyladenosine (m6A) modification as the most prevalent mammalian RNA internal modification has been considered as the invasive biomarkers in clinical diagnosis and biological mechanism researches. It is still challenged to explore m6A functions due to technical limitations on base- and location-resolved m6A modification. Herein, we firstly proposed a sequence-spot bispecific photoelectrochemical (PEC) strategy based on in situ hybridization mediated proximity ligation assay for m6A RNA characterization with high sensitivity and accuracy. Firstly, the target m6A methylated RNA could be transferred to the exposed cohesive terminus of H1 based on the special self-designed auxiliary proximity ligation assay (PLA) with sequence-spot bispecific recognition. The exposed cohesive terminus of H1 could furtherly trigger the next catalytic hairpin assembly (CHA) amplification and in situ exponential nonlinear hyperbranched hybridization chain reaction for highly sensitive monitoring of m6A methylated RNA. Compared with conventional technologies, the proposed sequence-spot bispecific PEC strategy for m6A methylation of special RNA based on proximity ligation-triggered in situ nHCR performed improved sensitivity and selectivity with a detection limit of 53 fM, providing new insights into highly sensitive monitoring m6A methylation of RNA in bioassay, disease diagnosis and RNA mechanism.

The Regulatory Network of METTL3 in the Nervous System: Diagnostic Biomarkers and Therapeutic Targets.

Methyltransferase-like 3 (METTL3) is a typical component of N6-methyladenosine writers that exhibits methyltransferase activity and deposits methyl groups on RNA. Currently, accumulating studies have demonstrated the involvement of METTL3 in the regulation of neuro-physiological and pathological events. However, no reviews have comprehensively summarized and analyzed the roles and mechanisms of METTL3 in these events. Herein, we are focused on reviewing the roles of METTL3 in regulating normal neurophysiological (Neurogenesis, Synaptic Plasticity and Glial Plasticity, Neurodevelopment, Learning and Memory,) and neuropathological (Autism Spectrum Disorder, Major Depressive Disorder, Neurodegenerative disorders, Brain Tumors, Brain Injuries, and Other Brain Disorders) events. Our review found that although the down-regulated levels of METTL3 function through different roles and mechanisms in the nervous system, it primarily inactivates neuro-physiological events and triggers or worsens neuropathological events. In addition, our review suggests that METTL3 could be used as a diagnostic biomarker and therapeutic target in the nervous system. Collectively, our review has provided an up-to-date research outline of METTL3 in the nervous system. In addition, the regulatory network for METTL3 in the nervous system has been mapped, which could provide directions for future research, biomarkers for clinical diagnosis, and targets for disease treatment. Furthermore, this review has provided a comprehensive view, which could improve our understanding of METTL3 functions in the nervous system.

Integrated analysis of circulating cell free nucleic acids for cancer genotyping and immune phenotyping of tumor microenvironment.

The circulating cell-free nucleic acids (ccfNAs) consist of a heterogenous cocktail of both single (ssNA) and double-stranded (dsNA) nucleic acids. These ccfNAs are secreted into the blood circulation by both healthy and malignant cells via various mechanisms including apoptosis, necrosis, and active secretion. The major source of ccfNAs are the cells of hematopoietic system under healthy conditions. These ccfNAs include fragmented circulating cell free DNA (ccfDNA), coding or messenger RNA (mRNA), long non-coding RNA (lncRNA), microRNA (miRNA), and mitochondrial DNA/RNA (mtDNA and mtRNA), that serve as prospective biomarkers in assessment of various clinical conditions. For, e.g., free fetal DNA and RNA migrate into the maternal plasma, whereas circulating tumor DNA (ctDNA) has clinical relevance in diagnostic, prognostic, therapeutic targeting, and disease progression monitoring to improve precision medicine in cancer. The epigenetic modifications of ccfDNA as well as circulating cell-free RNA (ccfRNA) such as miRNA and lncRNA show disease-related variations and hold potential as epigenetic biomarkers. The messenger RNA present in the circulation or the circulating cell free mRNA (ccf-mRNA) and long non-coding RNA (ccf-lncRNA) have gradually become substantial in liquid biopsy by acting as effective biomarkers to assess various aspects of disease diagnosis and prognosis. Conversely, the simultaneous characterization of coding and non-coding RNAs in human biofluids still poses a significant hurdle. Moreover, a comprehensive assessment of ccfRNA that may reflect the tumor microenvironment is being explored. In this review, we focus on the novel approaches for exploring ccfDNA and ccfRNAs, specifically ccf-mRNA as biomarkers in clinical diagnosis and prognosis of cancer. Integrating the detection of circulating tumor DNA (ctDNA) for cancer genotyping in conjunction with ccfRNA both quantitatively and qualitatively, may potentially hold immense promise towards precision medicine. The current challenges and future directions in deciphering the complexity of cancer networks based on the dynamic state of ccfNAs will be discussed.

Abstract 1049: Liquid biopsy-based comprehensive genomic profiling reveal mutational landscape in advanced breast cancer

Abstract Introduction: Breast cancer is the most common malignancy worldwide in females, with more than 2.26 million new cases and 680 thousand of deaths in 2020. Advanced breast cancer is characterized by complicated genomic landscape and requires comprehensive genomic profiling. However, tissue biopsy is hard to get in patients with recurrent metastasis. Meanwhile, limited studies have been reported to use liquid biopsy and were not capable of reporting gene copy number loss. Here we reported a comprehensive genomic profiling study in advanced breast cancer patients using liquid biopsy. Methods: The prospective study is part of the Predicine’s Phoenix Program, a global molecular biomarkers screening program in multiple solid tumors. Currently, the study enrolled 220 advanced breast cancer patients from 8 centers in China, who were naïve to the 1st line treatment. 10ml of blood was collected from each patient and delivered to a central lab for ctDNA analysis. The study applied PredicineCARE, an NGS-based liquid biopsy assay, to profile somatic mutations, copy number variations, and gene fusions among these patients. Results: The study identified 793 somatic mutations and 283 copy number variants among blood samples from 220 patients. The most common altered genes were TP53(101/220, 46%), PIK3CA(77/220, 35%), BRCA2(37/352, 17%), PTEN(26/220, 12%), and ATM(24/220, 11%). Gene copy number gain incidents were identified on MYC(n=28), AKT3(n=23), FGFR1(n=18), and PIK3CA(n=16), etc. The study also reported gene copy number loss incidents through liquid biopsy, such as CDKN2A(n=17), BRCA1(n=12), TP53(n=11), RB1(n=11), BRCA2(n=10), PTEN(n=9), and ATM(n=8). Conclusions: This study revealed the comprehensive mutational landscape of advanced breast cancer patients through liquid biopsy, providing novel biomarkers for clinical diagnosis and targeted therapy mechanism studies. Citation Format: Haoran Tang, Feng Xie, Yue Zhang, Shidong Jia. Liquid biopsy-based comprehensive genomic profiling reveal mutational landscape in advanced breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1049.

Photothermal visual sensing of alkaline phosphatase based on the etching of Au@MnO2 core-shell nanoparticles

Alkaline phosphatase (ALP), as a crucial enzyme involved in many physiological activities, is always used as one of the significant biomarkers in clinical diagnosis. Herein, a novel, simple, and effective photothermal quantitative method based on the etching of MnO2-coated gold nanoparticles (Au@MnO2 NPs) was established for ALP activity assay with a household thermometer-based visual readout. The photothermal effect of Au@MnO2 NPs is much higher than that of MnO2 NPs or Au NPs. The MnO2 shell of Au@MnO2 NPs can be etched by ascorbic acid, a product of ALP-catalyzed hydrolysis of 2-phospho-l-ascorbic acid. With the etching of Au@MnO2 NPs, the photothermal conversion efficiency decreased gradually, causing the decrease of the temperature increment of the solutions by degrees. A household thermometer, instead of large-scale and professional instruments, was used as a signal reader to realize the visual quantitative detection. The photothermal platform was used successfully for the determination of ALP with a wide linear range from 2.0 to 50 U/L and a detection limit as low as 0.75 U/L. Moreover, the inhibition efficiency of sodium vanadate for ALP activity was investigated, proving the photothermal quantitative method will be a potential platform for screening enzyme inhibitors. Such a sensitive, facile, and low-cost sensing assay provides a new prospect to develop platforms for point-of-care testing.